Transducer Mechanisms

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Chapter: Essential pharmacology : Pharmacodynamics Mechanism Of Drug Action; Receptor Pharmacology

Considerable progress has been made in the understanding of transducer mechanisms which in most instances have been found to be highly complex multistep processes that provide for amplification and integration of concurrently received extra and intracellular signals at each step.



Considerable progress has been made in the understanding of transducer mechanisms which in most instances have been found to be highly complex multistep processes that provide for amplification and integration of concurrently received extra and intracellular signals at each step. Because only a handful of transducer pathways are shared by a large number of receptors, the cell is able to generate an integrated response reflecting the sum total of diverse signal input. The transducer mechanisms can be grouped into 4 major categories. Receptors falling in one category have also been found to possess considerable structural homology, and belong to one super family of receptors.


1. Gprotein Coupled Receptors (GPCR)


These are a large family of cell membrane receptors which are linked to the effector (enzyme/ channel/carrier protein) through one or more GTPactivated proteins (Gproteins) for response effectuation. All such receptors have a common pattern of structural organization (Fig. 4.5). The molecule has 7 αhelical membrane spanning hydrophobic amino acid (AA) segments which run into 3 extracellular and 3 intracellular loops. The agonist binding site is located somewhere between the helices on the extracellular face, while another recognition site formed by cytosolic segments binds the coupling Gprotein. The Gproteins float in the membrane with their exposed domain lying in the cytosol, and are heterotrimeric in composition (α, β and γ subunits). In the inactive state GDP is bound to their exposed domain; activation through the receptor leads to displacement of GDP by GTP. The active αsubunit carrying GTP dissociates from the other two subunits and either activates or inhibits the effector. The βγ subunits have also been shown to modulate certain effectors like receptoroperated K+ channels, adenylylcyclase (AC) and phospholipase C.



A number of G proteins distinguished by their α subunits have been described. The important ones with their action on the effector are:


Gs     :         Adenylyl cyclase ↑, Ca2+ channel ↑

Gi      :         Adenylyl cyclase ↓, K+ channel ↑

Go     :         Ca2+ channel ↓

Gq     :         Phospholipase C ↑

G13   :         Na+/H+ exchange ↑


In addition Gn, Gk, Gt and Golf have been distinguished. A limited number of Gproteins are shared between different receptors and one receptor can utilize more than one Gprotein (agonist pleotropy), e.g. the following couplers have been associated with different receptors.


Receptor     Coupler


Muscarinic  Gi, Go, Gq

Dopamine D2       Gi, Go

βadrenergic Gs, Gi

α1adrenergic         Gq

α2adrenergic         Gi, Gs, Go

GABAB              Gi, Go

5HT          Gi, Gq, Gs, Gk


In addition, a receptor can utilize different biochemical pathways in different tissues.


The α-subunit has GTPase activity: the bound GTP is slowly hydrolysed to GDP: the αsubunit then dissociates from the effector to rejoin its other subunits, but not before the effector has been activated/inhibited for a few seconds and the signal has been amplified. The onset time of response through this type of receptors is also in seconds.


There are three major effector pathways (Table 4.1) through which GPCRs function.


a) Adenylyl cyclase: cAMP pathway Activation of AC results in intracellular accumulation of second messenger cAMP (Fig. 4.6) which functions mainly through cAMPdependent protein kinase (PKA). The PKA phosphorylates and alters the function of many enzymes, ion channels, transporters and structural proteins to manifest as increased contractility/impulse generation (heart), relaxation (smooth muscle), glycogenolysis, lipolysis, inhibition of secretion/mediator release, modulation of junctional transmission, hormone synthesis, etc. In addition, cAMP directly opens a specific type of membrane Ca2+ channel called cyclic nucleotide gated channel (CNG) in the heart, brain and kidney. Responses opposite to the above are produced when AC is inhibited through inhibitory G-protein.


b) Phospholipase C: IP3DAG pathway Activation of phospholipase C (PLc) hydrolyses the membrane phospholipid phosphatidyl inositol 4, 5bisphosphate (PIP2) to generate the second messengers inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG). The IP3 mobilises Ca2+ from intracellular organellar depots and DAG enhances protein kinase C (PKc) activation by Ca2+ (Fig. 4.7). Cytosolic Ca2+ (third messenger in this setting) is a highly versatile regulator acting through calmodulin (CAM), PKc and other effectors—mediates/modulates contraction, secretion/transmitter release, eicosanoid synthesis, neuronal excitability, intracellular movements, membrane function, metabolism, cell proliferation, etc. Like AC, the PLc can also be inhibited through inhibitory Gprotein when directionally opposite responses would be expected.



Intracellular Ca2+ release has been found to occur in waves (Ca2+ mediated Ca2+ release from successive pools facilitated by inositol 1, 3, 4, 5tetrakisphosphate—IP4) and exhibits a variety of agonist and concentration dependent oscillatory patterns. The activation of different effectors may depend on the amplitude and pattern of these oscillations. Thus, the same intracellular messenger can trigger different responses depending on the nature and strength of the extracellular signal.


          c) Channel regulation The activated Gproteins can also open or close ionic channels specific for Ca2+, K+ or Na+, without the intervention of any second messenger like cAMP or IP3, and bring about hyperpolarization/depolarization/ changes in intracellular Ca2+. The Gs opens Ca2+ channels in myocardium and skeletal muscles, while Gi and Go open K+ channels in heart and smooth muscle as well as close neuronal Ca2+ channels. Physiological responses like changes in inotropy, chronotropy, transmitter release, neuronal activity and smooth muscle relaxation follow. Receptors found to regulate ionic channels through Gproteins are listed in Table 4.1.




2. Receptors With Intrinsic Ion Channel


These cell surface receptors, also called ligand gated ion channels, enclose ion selective channels (for Na+, K+, Ca2+ or Cl¯) within their molecules. Agonist binding opens the channel (Fig. 4.4) and causes depolarization/hyperpolarization/ changes in cytosolic ionic composition, depending on the ion that flows through. The nicotinic cholinergic, GABAA, glycine (inhibitory), excitatory AA (kainate, NMDA or NmethylDaspartate, quisqualate) and 5HT3 receptors fall in this category.


The receptor is usually a pentameric protein; all subunits, in addition to large intra and extracellular segments, generally have four membrane spanning domains in each of which the AA chain traverses the width of the membrane six times. The subunits are thought to be arranged round the channel like a rosette and the α subunits usually bear the agonist binding sites.


Certain receptoroperated (or ligandgated) ion channels also have secondary ligands which bind to an allosteric site and modulate the gating of the channel by the primary ligand, e.g. the benzodiazepine receptor modulates GABAA gated Cl¯channel.


Thus, in these receptors, agonists directly operate ion channels, without the intervention of any coupling protein or second messenger. The onset and offset of responses through this class of receptors is the fastest (in milliseconds).


3. Enzyme-Linked Receptors


This class of receptors have a subunit with enzymatic property or bind a JAK (Janus Kinase) enzyme on activation. The agonist binding site and the catalytic site lie respectively on the outer and inner face of the plasma membrane (Fig. 4.8). These two domains are interconnected through a single transmembrane stretch of peptide chain. There are two major subgroups of such receptors.


a. Those that have intrinsic enzymatic activity.



b. Those that lack intrinsic enzymatic activity, but bind a JAKSTAT kinase on activation.



a.     Intrinsic Enzyme Receptors


The intracellular domain is either a protein kinase or guanylyl cyclase.

In most cases the protein kinase specifically phosphorylates tyrosine residues on substrate proteins (Fig. 4.8A), e.g. insulin, epidermal growth factor (EGF), nerve growth factor (NGF) receptors, but in few it is a serine or threonine protein kinase. In the monomeric state, the kinase remains inactive. Agonist binding induces dimerization of receptor molecules and activates the kinase to autophosphorylate tyrosine residues on each other, increasing their affinity for binding substrate proteins and carrying forward the cascade of tyrosine phosphorylations. Intracellular events are triggered by phosphorylation of relevant proteins, many of which carry a SH2 domain. A general feature of this class of receptors is that their dimerization also promotes receptor internalization, degradation in lysosomes and down regulation.


The enzyme can also be guanylyl cyclase (GC), as in the case of atrial natriuretic peptide (ANP). Agonist activation of the receptor generates cGMP in the cytosol as a second messenger which in turn activates cGMPdependent protein kinase and modulates cellular activity.



b. JAK-STAT Kinase Binding Receptors


These receptors differ in not having any intrinsic catalytic domain. Agonist induced dimerization alters the intracellular domain conformation to increase its affinity for a cytosolic tyrosine protein kinase JAK. On binding, JAK gets activated and phosphorylates tyrosine residues of the receptor, which now binds another free moving protein STAT (signal transducer and activator of transcription) which is also phosphorylated by JAK. Pairs of phosphorylated STAT dimerize and translocate to the nucleus to regulate gene transcription resulting in a biological response. Many cytokines, growth hormone, interferons, etc. act through this type of receptor.


The enzymelinked receptors transduce responses in a matter of few minutes to a few hours.


4. Receptors Regulating Gene Expression (Transcription Factors)


In contrast to the above 3 classes of receptors, these are intracellular (cytoplasmic or nuclear) soluble proteins which respond to lipid soluble chemical messengers that penetrate the cell (Fig. 4.9). The receptor protein (specific for each hormone/ regulator) is inherently capable of binding to specific genes, but is kept inhibited till the hormone binds near its carboxy terminus and exposes the DNA binding regulatory segment located in the middle of the molecule. Attachment of the receptor protein to the genes facilitates their expression so that specific mRNA is synthesized on the template of the gene. This mRNA moves to the ribosomes and directs synthesis of specific proteins which regulate the activity of target cells.


All steroidal hormones (glucocorticoids, mineralocorticoids, androgens, estrogens, progesterone), thyroxine, vit D and vit A function in this manner. Different steroidal hormones affect different target cells and produce different effects because each one binds to its own receptor and directs a unique pattern of synthesis of specific proteins. The specificity as to which hormone will be bound is provided by the hormone binding domain, while that as to which gene will be activated or repressed is a function of the DNA binding/Nterminus domain. Chimeric receptors have been produced which respond to one hormone, but produce the effects of the other hormone.


This transduction mechanism is the slowest in its time course of action (takes hours).


Receptor Regulation


Receptors exist in a dynamic state; their density and efficacy is subject to regulation by the level of on going activity, feedback from their own signal output and other physio-pathological influences. In tonically active systems, prolonged deprivation of the agonist (by denervation or continued use of an antagonist or a drug which reduces input) results in super sensitivity of the receptor as well as the effector system to the agonist. This has clinical relevance in clonidine/CNS depressant/ opioid withdrawal syndromes, sudden discontinuation of propranolol in angina pectoris, etc. The mechanisms involved may be unmasking of receptors or their proliferation (up regulation) or accentuation of signal amplification by the transducer.


Conversely, continued/intense receptor stimulation causes desensitization or refractoriness: the receptor becomes less sensitive to the agonist. This can be easily demonstrated experimentally (Fig. 4.10); clinical examples are bronchial asthma patients treated continuously with β adrenergic agonists and parkinsonian patients treated with high doses of levodopa. The changes may be brought about by:

i) Masking or internalization of the receptor (it becomes inaccessible to the agonist). In this case refractoriness develops as well as fades quickly.


In the case of β adrenergic receptor, it has been found that agonist binding promotes phosphorylation of its serine residues near the intracellular carboxy terminus by an enzyme β adrenergic receptor kinase (βARK), allowing it to bind a protein called βarrestin which hinders its interaction with Gs receptor transduction is impaired. When the βagonist is removed, the serine residues are dephosphorylated and receptor mediated activation of Gs is restored.


ii) Decreased synthesis/increased destruction of the receptor (down regulation): refractoriness develops over weeks or months and recedes slowly. Receptor down regulation is particularly exhibited by the tyrosine protein kinase receptors.


Some times response to all agonists which act through different receptors but produce the same overt effect (e.g. histamine and acetylcholine both contract intestinal smooth muscle) is decreased by exposure to any one of these agonists (heterologous desensitization), showing that mechanisms of response effectuation have become less efficient. However, often desensitization is limited to agonists of the receptor being repeatedly activated (homologous desensitization).


Both homologous and heterologous desensitization has been observed in the case of GPCRs. The BARKβ arrestin mechanism described above produces homologous desensitization. The GPCRs transduce many responses by activating PKA and PKC. These kinases phosphorylate the GPCRs as well rather non-selectively (at a site different from that of BARK) and hinder their interaction with G-proteins, resulting in heterologous desensitization.


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